Production of Protein Hydrolysates from Cod Backbone Using Selected Enzymes: Evaluation of Antioxidative and Antimicrobial Activities of Hydrolysates

In the fish industry, up to 70% of all fish end up as side-streams such as backbones, heads, and viscera. To reduce the quantities of side-streams, a higher utilization degree of fish is needed. The aim of this study was to use cod backbone for an enzymatic production of bioactive hydrolysates with antioxidative and/or antimicrobial properties. Three different enzymes were applied (Alcalase, Neutrase, and Protamex), and hydrolyses were carried out within the enzyme's optima for pH and temperature for 0.5-6 h. The efficiency of the enzyme treatment was evaluated based on the protein extraction yield (PEY), the degree of hydrolysis (DH), and antioxidant activity using two different in vitro assays (1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and iron chelation) and antimicrobial activity determined by minimum inhibitory concentration (MIC) and disk diffusion assays. Selected hydrolysates showing activity were evaluated with respect to amino acid composition and molecular weight. Alcalase-treated samples had the highest PEY (3 h, 63.5 ± 4.5%) followed by Protamex-treated samples (3 and 6 h; 51.9 ± 5.5% and 56.5 ± 4.5%); the lowest PEY was obtained with Neutrase (3 and 6 h; 30.4 ± 1.9% and 34.7 ± 3.4%). No clear relationship was observed between the PEY and DH. All hydrolysates had antioxidant activities. For radical scavenging activity, Protamex-treated hydrolysate showed the lowest IC₅₀ (6 h, 2.1 ± 0.1 mg powder/mL) and had a molecular weight <10 kDa, whereas for iron chelation activity, the control samples (no enzyme added but heat-treated) showed a similar or lower IC₅₀ with molecular weights of 200-10 kDa. Amino acid composition measured on selected hydrolysates suggested that not only the composition of amino acid but also sequence and size influence the properties. None of the hydrolysates showed antimicrobial activity. In summary, the results showed that protein hydrolysates with antioxidant activity can be produced from the cod backbone, which makes it possible to utilize this side-stream generated in the fish industry.